PC-AssaySubmit ::= {
assay descr {
aid {
id 652264,
version 1
},
aid-source db {
name "NCGC",
source-id str "USP2607",
date std {
year 2014,
month 1,
day 16
}
},
name "qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2
as the Reporter: Cell Cycle",
description {
"Homeostasis of cellular proteins is maintained through a combination of
synthesis and degradation. The pathway that accounts for the majority of
protein degradation is the ubiquitin-proteasomal pathway. Ubiquitin (Ub) is
highly conserved in all cells and the generation of a multi-Ub chain
typically targets proteins for degradation by the proteasome. However,
ubiquitination is highly reversible and dynamic. Deubiquitination, the
reverse process, is catalyzed through the action of enzymes referred to as
isopeptidases or deubiquitinating enzymes (DUBs) [1, 2]. This group of
enzymes is collectively responsible for maintaining adequate pools of free
ubiquitin and regulating the ubiquitination status of cellular proteins. The
class of DUBs referred to as the ubiquitin-specific proteases (USP) family
functions endoproteolytically to cleave Ub chains from a wide range of
protein substrates. USP2a deubiquitinates fatty acid synthase (FASN) which
has recently been identified as an emerging oncology target. To identify
inhibitors of USP2a a cell-free qHTS assay was employed.",
"",
"Selected active compounds with corresponding inactive controls were
tested for their effect on the cell cycle in HCT116 cells. The USP2
substrate cyclin D1 is important for progression for G1 to S in the cell
cycle. An inhibitor of USP2 would lead to a decrease in the amount of cyclin
D1 and would arrest the cells in G1. Because the cells are predominantly in
G1 in the absence of compound treatment, nocodazole (a known G2 blocker) was
used to trap cells that made it through G1. Cell cycle was visualized using
PI staining. Cells in G2 have twice the fluorescence as cells in G1, and the
Acumen allows the cells to be binned by their individual fluorescence
properties. Therefore, in the presence of an active USP2 inhibitor the
fluorescence of a cell would be lower since it would not progress from G1 to
S and then G2. The cells were cultured in DMEM with 10% FBS and 1% Pen-Strep
and plated in black clear bottom collagen-coated 96-well plates",
"",
"NIH Molecular Libraries Probe Centers Network [MLPCN]",
"NIH Chemical Genomics Center [NCGC]",
"",
"MLPCN Grant: MH079852",
"PI Name: Benjamin Nicholson, Progenra Inc, Malvern, PA"
},
protocol {
"HCT116 cells at 600 cells/well were plated in 96-well plates and
allowed to adhere overnight (37C, 50% CO2). The following day media
containing a fixed concentration of nocodazole (final concentration of 0.6nM)
and serial dilutions of the test compounds (highest concentration at 39 uM)
was added to the cells. The cells were incubated for 16 hours. The cells were
fixed with 8% paraformaldehyde for 1 hour at room temperature and washed 3x
with 100 uL PBS/0.02% Triton solution. The cells were permeabilized with
PBS/0.5% Triton-X for 10 min. Twenty five microliter of Propidium iodide /
RNase reagent was added and the plate was incubated at 37C for 1 hour.
Intensity of the DNA stain per cell was quantified using the Acumen plate
reader. A cell cycle histogram report was generated to assess the effects of
the compound on cell cycle."
},
comment {
"If a compound has an IC50 <=5 uM, the compound is considered ""active""
and a score of 90 is assigned; if a compound has an IC50 > 5 uM and < 10 uM
then the compound is considered ""inconclusive"" and a score of 50 is
assigned; if a compound has an IC50 >= 10 uM it is considered ""inactive""
and a score of 10 is assigned."
},
xref {
{
xref aid 2281
},
{
xref taxonomy 9606
},
{
xref protein-gi 188528692
},
{
xref gene 9099
}
},
results {
{
tid 1,
name "IC50 (uM)",
type float,
unit um
}
},
revision 2,
activity-outcome-method other,
grant-number {
"MH079852"
},
project-category mlpcn
},
data {
{
sid 162009865,
version 1,
outcome active,
rank 90,
data {
{
tid 1,
value fval { 2, 10, 0 }
}
},
date std {
year 2013,
month 4,
day 3
}
}
}
}